Project Summary Among the commercially available systems for single cell isolation and next generation sequencing (NGS) sample preparation, none are capable of automating both imaging and NGS sample preparation at high throughput. While single cell genomic analysis has improved in sensitivity and throughput, concomitant improvements in detailed phenotypic characterization of cells has not been integrated into automated workflows, nor have they been scaled beyond a few dozen cells per run. Currently, the only means of biomarker- based sorting of cells prior to single cell sequencing is either fluorescence-activated cell sorting (FACS) or imaging cells on a microscope which is separate from an automated sample preparation instrument (e.g. Fluidigm C1, WaferGen ICELL8). For example, 10X Genomics? technology relies on upfront FACS purification of cell populations, and still cannot connect these relatively superficial phenotypic observations to downstream NGS data. Fluidigm?s C1 system allows imaging of the approximately 800 cells loaded on a microfluidic device on a separate microscope, but this method results in significant rates of multiplets and requires investigators to construct their own imaging methods, equipment and software, independent of the C1 system. Fluidigm?s Polaris allows both imaging and NGS sample preparation in one instrument but is limited in throughput to 48 cells per run and represents a significant capital expense. To address the unmet need for integrated imaging and NGS sample preparation in a single, high-throughput, cost-effective system, Cell Microsystems proposes here the development of the AIR-FLOW? System. Using our core CellRaft Technology, the AIR-FLOW? will allow multi-channel fluorescent imaging, isolation of single cells and RNA-Seq library preparation. The AIR-FLOW? System will allow multi-channel automated imaging of surface markers, morphology and even subcellular features, while integrating a microfluidic sample preparation method developed by Peter Sims, PhD of Columbia University to construct next generation sequencing (NGS) transcriptomic libraries. Dr. Sims? sample preparation technology is highly complementary with the CellRaft technology: both rely on microwell arrays, fluorescence imaging and the same biocompatible materials. This approach employs optically barcoded beads for mRNA capture and sample preparation, allowing the resulting sequencing data to be directly linked to imaging data on a cell-to-cell basis. Also, by integrating these two technologies, throughputs of several thousand cells per 4-5 hr run will be easily achievable. Based on our Phase I data, Cell Microsystems? core CellRaft Technology, provides key advantages over existing microfluidic technologies, effectively eliminating cell-to-cell cross-contamination, reducing sample input requirements and providing a less stressful environment which reduces transcriptomic artifacts. During this Phase II program, we will integrate Dr. Sims? technology with the core CellRaft Technology in the AIR-FLOW? System.